plants Article Integrating DNA Barcoding and Traditional Taxonomy for the Identification of Dipterocarps in Remnant Lowland Forests of Sumatra Carina Carneiro de Melo Moura 1 , Fabian Brambach 2 , Kevin Jair Hernandez Bado 1, Konstantin V. Krutovsky 1,3,4,5,6 , Holger Kreft 2, Sri Sudarmiyati Tjitrosoedirdjo 7, Iskandar Z. Siregar 8 and Oliver Gailing 1,3,* 1 Department of Forest Genetics and Forest Tree Breeding, University of Göttingen, Büsgenweg 2, 37077 Göttingen, Germany; [email protected] (C.C.d.M.M.); [email protected] (K.J.H.B.); [email protected] (K.V.K.) 2 Biodiversity, Macroecology and Biogeography, University of Göttingen, Büsgenweg 1, 37077 Göttingen, Germany; [email protected] (F.B.); [email protected] (H.K.) 3 Center for Integrated Breeding Research, University of Göttingen, Albrecht-Thaer-Weg 3, 37075 Göttingen, Germany 4 Laboratory of Population Genetics, N. I. Vavilov Institute of General Genetics, Russian Academy of Sciences, 3 Gubkin Str., Moscow 119333, Russian 5 Laboratory of Forest Genomics, Genome Research and Education Center, Institute of Fundamental Biology and Biotechnology, Siberian Federal University, 50a/2 Akademgorodok, Krasnoyarsk 660036, Russia 6 Department of Ecosystem Science and Management, Texas A&M University, College Station, TX 77843-2138, USA 7 Southeast Asian Regional Center for Tropical Biology (SEAMEO BIOTROP), Jalan Raya Tajur Km. 6, Bogor 16144, Indonesia; [email protected] 8 Department of Silviculture, Faculty of Forestry, Bogor Agricultural University, Dramaga Campus, Bogor 16680, Indonesia; [email protected] * Correspondence: [email protected]; Tel.: +49-551-3933536 Received: 23 August 2019; Accepted: 17 October 2019; Published: 30 October 2019 Abstract: DNA barcoding has been used as a universal tool for phylogenetic inferences and diversity assessments, especially in poorly studied species and regions. The aim of this study was to contrast morphological taxonomy and DNA barcoding, using the three frequently used markers matK, rbcL, and trnL-F, to assess the efficiency of DNA barcoding in the identification of dipterocarps in Sumatra, Indonesia. The chloroplast gene matK was the most polymorphic among these three markers with an average interspecific genetic distance of 0.020. The results of the molecular data were mostly in agreement with the morphological identification for the clades of Anthoshorea, Hopea, Richetia, Parashorea, and Anisoptera, nonetheless these markers were inefficient to resolve the relationships within the Rubroshorea group. The maximum likelihood and Bayesian inference phylogenies identified Shorea as a paraphyletic genus, Anthoshorea appeared as sister to Hopea, and Richetia was sister to Parashorea. A better discriminatory power among dipterocarp species provided by matK and observed in our study suggests that this marker has a higher evolutionary rate than the other two markers tested. However, a combination of several different barcoding markers is essential for reliable identification of the species at a lower taxonomic level. Keywords: matK; rbcL; trnL-F; Dipterocarpoideae; tropical tree diversity; genetic distance; reference DNA library Plants 2019, 8, 461; doi:10.3390/plants8110461 www.mdpi.com/journal/plants Plants 2019, 8, 461 2 of 14 1. Introduction The Dipterocarpaceae family consists of approximately 680 species that are commonly placed in the two subfamilies, Monotoideae (~30 species in Africa and Madagascar, and the monotypic Pseudomonotes in Colombia) and Dipterocarpoideae, which hold the majority of species and are confined to the eastern tropics from India to New Guinea [1–6]. Members of the Dipterocarpoideae dominate the diverse rainforests of Sundaland, often with many co-occurring species [7]. Due to their high abundance, mechanical wood-properties, and tall stature, they provide some of the most valued and abundant sources of tropical hardwood, and at the same time are significant stores of aboveground carbon [8]. Hence, dipterocarps are of great ecological and economic importance [6], and understanding their diversity is necessary to advance our general knowledge of Southeast Asian rainforests. Despite being some of the most prevalent trees in the Asian tropics, dipterocarps are increasingly threatened by deforestation and land-use change [9]. For effective conservation of the threatened species and genetic diversity of the Dipterocarpaceae, it is important to understand their species-level taxonomy as well as their origin and the evolutionary processes that have led to the astonishing diversity of the family. To this end, molecular phylogenies are needed that provide basic knowledge on the evolutionary history and phylogenetic relationships of extant species [10]. Indeed, the dipterocarp family has already received great interest in traditional morphology-based taxonomy [11–13] and different phylogenetic studies based on DNA-markers ranging from Restriction Fragment Length Polymorphism (RFLP) of plastid regions [1], plastid DNA sequences [14,15], internal transcribed spacers [16], and nuclear genes [17] to genome-wide markers [3,18,19]. The exact taxonomic placement of the Dipterocarpaceae family within the angiosperms was disputed for a long time, including placements into the orders Theales and Malvales [20]. Based on recent phylogenetic and phylogenomic studies, the placement in Malvales is now universally accepted, but the relationships among the Dipterocarpaceae and the closely related families Cistaceae and Sarcolaenaceae are still insufficiently resolved [21]. The largest tribe of Dipterocarpaceae, Shoreeae, consists of the genera Shorea, Hopea, Parashorea, and the monotypic Neobalanocarpus [22]. Generic limits in the tribe are obscure and recent studies have shown that Shorea is in fact paraphyletic with Hopea, Parashorea, and Neobalanocarpus nested within it [3,23]. Nevertheless, based on phylogenomic data, well-defined clades within the tribe Shoreeae can be identified. They are consistent with most of the traditional genera and recognized subgenera of Shorea: Anthoshorea (white meranti), Richetia (yellow meranti), Shorea (balau/selangan batu), Rubroshorea (red meranti), and Doona (Pentacme has not been included in phylogenomic studies so far) [3,19]. Despite the comparatively large attention that this group has received due to its economic importance, identification of dipterocarps can be challenging [11–13]. This is due mainly to the large size of most dipterocarps and their characteristic periodic mass flowering and fruiting, which results in trees with reproductive structures absent for most of the time. Another complicating factor—especially in ecological studies—is that several closely related and morphologically similar species may co-occur. In this context, DNA barcoding can provide an independent source of information to delimit and identify species [24,25]. The matK and rbcL loci are considered standard plant DNA barcoding markers due to their universality, relatively high overall sequence quality, low cost, and high discriminatory power between angiosperms [26,27]. Specifically, rbcL has a higher PCR amplification success, but lower discriminatory power than matK. The use of both barcoding markers, rbcL together with matK, was proposed by the CBOL Plant Working Group of the Consortium for the Barcoding of Life [28] to have a higher combined discriminatory power [29]. Non-coding regions have also been implemented as barcoding marker [30]. Thus, joint use of coding and non-coding regions is an important step to implement a plant barcode database as a tool for accurate diversity assessments and to develop conservation strategies. A detailed DNA barcoding dataset of dipterocarps with comprehensive coverage across taxonomic groups and geographic areas is currently lacking [25]. So far, most efforts have concentrated on the Malay Peninsula and Borneo [3,16,24], while material from Sumatra has hardly been included in Plants 2019, 8, 461 3 of 14 analyses (but see [25]). At the same time, large-scale logging and subsequent deforestation in Sumatra over the last decades [31] have decimated dipterocarp populations on the island, and many of the formerly widespread species are now threatened with extinction [32]. Detailed assessments of dipterocarp diversity and composition in the remaining forests of Sumatra are therefore a requirement for effective conservation measures. Here, we contrast the traditional morphological taxonomy and the DNA barcoding approach for the identification of dipterocarp species in remnant lowland rainforests of Sumatra, Indonesia. We used three DNA barcoding markers—rbcL, matK, and trnL-F to (i) assess the dipterocarp identification using phylogenetic trees; and (ii) test the efficiency of these markers for the identification of dipterocarps. 2. Materials and Methods 2.1. Study Area and Specimen Collection The study was a part of the Collaborative Research Centre 990: Ecological and Socio-economic Functions of Tropical Lowland Rainforest Transformation Systems (CRC990: EFForTS project, https: //www.uni-goettingen.de/efforts) in Jambi Province, central Sumatra, Indonesia. The study region is characterized by an average annual temperature of 26.7 0.2 C and mean annual precipitation of ± ◦ approximately 2235 381 mm [25,33,34]. Samples were collected in two areas, the ‘Bukit Duabelas ± landscape’ and the ‘Harapan landscape’, respectively, as part of plot based (0.25 ha, 50 50 m, × four plots per landscape and land-use) inventories on well-drained